On 2017 Jun 10, Shawn McGlynn commented:

With the trees from this phylogeny paper now available, we can resolve the discussion between myself and the authors (below) and conclude that there is no evidence that nitrogenase was present in the LUCA as the authors claimed in their publication.

In their data set, the authors identified two clusters of proteins which they refer to as NifD; clusters 3058 and 3899. NifD binds the metal cluster of nitrogenase and is required for catalysis. In the author's protein groups, cluster 3058 is comprised of 30 sequences, and 3899 is comprised of 10 sequences. Inspection of these sequences reveals that neither cluster contains any actual NifD sequences. This can be said with certainty since biochemistry has demonstrated that the metal cofactor coordinating residues Cys²⁷⁵ and His⁴⁴² (using the numbering scheme from the Azotobacter vinelandii NifD sequence) are absolutely required for activity. NONE of the 40 sequences analyzed by the authors contain these residues. Therefore, NONE of these sequences can have the capability to bind the nitrogenase metal cluster, and it follows that none of them would have the capacity to reduce di-nitrogen. The authors have not analyzed a single nitrogenase sequence in their analysis and are therefore disqualified from making claims about the evolution of the protein; the claims made in this paper about nitrogenase cannot be substantiated with the data which have been analyzed. The sequences contained in the author's "NifD" protein clusters are closely related homologs related to nitrogenase cofactor biosynthesis and are within a large family of related proteins (which includes real NifD proteins, but also proteins involved in bacteriochlorophyll and Ni porphyrin F430 biosynthesis). While the author's analyzed proteins are more related to nitrogen metabolism than F430 or bacteriochlorophyll biosynthesis, they are not nitrogenase, but are nitrogenase homologs that complete assembly reactions.

Other than not having looked at any sequences which would be capable of catalyzing nitrogen reduction, the presentation of two "NifD" clusters highlights important problems with the methods used in this paper which affect the entire analysis and conclusions. First, two clusters were formed for one homologous group, which should not have occurred if the goal was to investigate ancestry. Second, by selecting small clusters from whole trees, the authors were able to prune the full tree until they recovered small sub trees which show monophyly of archaea and bacteria. However it was incorrect to ignore the entire tree of homologs and present only two small clusters from a large family. This is "cherry" picking to the extreme - in this case it is "nitrogenase" picking, but it is very likely that this problem of pruning until the desired result sullies many if not all of the protein families and conclusions in the paper; for example the radical SAM tree was likely pruned in this same way with the incorrect conclusion being reached (like nitrogenase, a full tree of radical SAM does not recover the archaea bacteria split in protein phylogenies either). Until someone does a complete analysis with full trees the claims of this paper will remain unproven and misleading since they are based on selective sampling of information. It would seem that the authors have missed the full trees whilst being lost in mere branches of their phylogenetic forest of 286,514 protein clusters.

In a forthcoming publication, I will discuss in detail the branching position of the NifD homologs identified by the authors, as well as the possible evolutionary trajectory of the whole protein family with respect to the evolution of life and the nitrogen cycle on this planet in more detail, including bona fide NifD proteins which I have already made comment on below in this PubMed Commons thread.

On 2017 Mar 20, Madeline C Weiss commented:

The trees and also the alignments for all 355 proteins are available on our resources website:

http://www.molevol.de/resources/index.html?id=007weiss/

On 2016 Dec 25, Shawn McGlynn commented:

Unfortunately, the points raised by Professor Martin do not address the problem I raised in my original comment, which I quote from below: "nitrogenase protein phylogeny does not recover the monophyly of the archaea and bacteria." As I wrote, the nitrogenase protein is an excellent example of violating the author's criterion of judging a protein to be present in the LUCA by virtue that "its tree should recover bacterial and archaeal monophyly" (quoted from Weiss et alia). Therefore it should not be included in this paper's conclusions.

Let's be more specific about this and look at a phylogenetic tree of the nitrogenase D peptide (sometimes referred to as the alpha subunit). This peptide binds the catalytic metal-sulfur cluster and its phylogeny can be viewed on my google site https://sites.google.com/site/simplyshawn/home.

I colored archaeal versions red and bacterial black. You can see that this tree does not recover the monophyly of the archaea and bacteria and therefore should not be included in the author's set of LUCA proteins.

Is what I display the result of a tree construction error? Probably not, this tree looks pretty much the same to every other tree published by various methods, so it seems to correctly reflect sequence evolution as we understand it today. The tree I made just has more sequences; it can be compared directly with Figure 1 in Leigh 2000, Figure 2 in Raymond et alia 2004, and Figure 2 in Boyd et alia 2011. Unfortunately, Weiss and others do not include any trees in their paper, so it is impossible to know what they are deriving their conclusions from, but it would be very difficult to imagine that they have constructed a tree different from all of these.

Could it be that all these archaea obtained nitrogenase by horizontal gene transfer after the enzyme emerging in bacteria? Possibly, although this would imply that it was not in the LUCA as the authors claim.

Could it be that the protein developed in methanogens and was then transferred into the bacterial domain? Yes, and Boyd and others suggested just this in their 2011 Geobiology paper. This would also mean that the protein was not in the LUCA.

Could it be that the protein was present in the LUCA as Weiss and co-authors assert? Based on phylogenetic analysis, no.

As Prof. Martin writes - there certainly is more debate to be had about nitrogenase age than was visible in my first comment. However, we can be sure that the protein does not recover the archaea bacteria monophyly, and should have not been included in the authors paper.

Prof. Martin might likely counter my arguments here by saying something about metal dependence and treating different sequences separately (for example Anf, Vnf, MoFe type). However let us remember that the sequences are all homologous. Metal binding is one component of the nitrogenase phenotype, but all nitrogenase are homologous and descend from a common ancestor.

Now that we can be sure that the nitrogenase does not conform to the author's second criterion for judging presence in the LUCA, let us examine if the protein conforms to the first criterion: "the protein should be present in at least two higher taxa of bacteria and archaea". In fact, all nitrogenase in archaea that are found in the NCBI and JGI databases are only within the methanogenic euryarchaeota. Unfortunately, Weiss and coauthors do not define what "higher taxa" means to them in their article, but it should be questioned if having a gene represented by members of a single phylum actually constitutes being present within "two higher taxa". Archaea are significantly more diverse than what is observed in the methanogenic euryarchaeota. Surely, if a protein was present in the LUCA, it would be a bit more widely distributed, and it would be easy to argue that the presence of nitrogenase in only one phylum provides evidence that it does not conform to the authors criterion number one. Thus, the picture that emerges from a closer look at nitrogenase phylogeny and distribution is that the protein violates both of the authors criteria for inclusion in the LUCA protein set.

Let me summarize:

1) Nitrogenase does not recover the bacterial and archaeal monophyly and therefore violates the author's criterion number 2.

2) Nitrogenase in archaea is only found within the methanogenic euryarchaeota and is not broadly distributed, and therefore also seems to violate the authors criterion number 1.

3) From a phylogenetic perspective, the nitrogenase protein should not be included as a candidate to be present in the LUCA.

On 2016 Oct 12, William F Martin commented:

There is an ongoing debate in the literature about the age of nitrogenase.

In his comment, McGlynn favours published interpretations that molybdenum nitrogenase arose some time after the Great Oxidation Event 2.5 billion years ago (1). A different perspective on the issue is provided by Stüecken et al. (2) who found evidence for Mo-nitrogenase before 3.2 billion years ago. Our recent paper (3) traced nitrogenase to LUCA, but also suggested that methanogens are the ancestral forms of archaea, in line both with phylogenetic (4) and isotope (5) evidence for the antiquity of methanogens, and with a methanogen origin of nitrogenase (6).

Clearly, there had to be a source of reduced nitrogen at life’s origin before the origin of nitrogenase or any other enzyme. Our data (3) are consistent with the view that life arose in hydrothermal vents and independent laboratory studies show that dinitrogen can be reduced to ammonium under simulated vent conditions (7,8). There is more to the debate about nitrogenase age, methanogen age, and early sources of fixed nitrogen than McGlynn’s comment would suggest.

1. Boyd, E. S., Hamilton, T. L., and Peters, J. W. (2011). An alternative path for the evolution of biological nitrogen fixation. Front. Microbiol. 2:205. doi:10.3389/fmicb.2011.00205

2. Stüeken EE, Buick R, Guy BM, Koehler MC. Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr. Nature 520, 666–669 (2015)

3. Weiss MC, Sousa FL, Mrnjavac N, Neukirchen S, Roettger M, Nelson-Sathi S, Martin WF: The physiology and habitat of the last universal common ancestor. Nat Microbiol (2016) 1(9):16116 doi:10.1038/nmicrobiol.2016.116

4. Raymann, K., Brochier-Armanet, C. & Gribaldo, S. The two-domain tree of life is linked to a new root for the Archaea. Proc. Natl Acad. Sci. USA 112, 6670–6675 (2015).

5. Ueno, Y., K. Yamada, N. Yoshida, S. Maruyama, and Y. Isozaki. 2006. Evidence from fluid inclusions for microbial methanogenesis in the early archaean era. Nature 440:516-519.

6. Boyd, E. S., Anbar, A. D., Miller, S., Hamilton, T. L., Lavin, M., and Peters, J. W. (2011). A late methanogen origin for molybdenum-depen- dent nitrogenase. Geobiology 9, 221–232.

7. Smirnov A, Hausner D, Laffers R, Strongin DR, Schoonen MAA. Abiotic ammonium formation in the presence of Ni-Fe metals and alloys and its implications for the Hadean nitrogen cycle. Geochemical Transactions 9:5 (2008) doi:10.1186/1467-4866-9-5

8. Dörr M, Kassbohrer J, Grunert R, Kreisel G, Brand WA, Werner RA, Geilmann H, Apfel C, Robl C, Weigand W: A possible prebiotic formation of ammonia from dinitrogen on iron sulfide surfaces. Angew Chem Int Ed Engl 2003, 42(13):1540-1543.

On 2017 Mar 09, Tanai Cardona commented:

I agree with Shawn regarding the fact that "Nitrogenase does not recover the bacterial and archaeal monophyly and therefore violates the author's criterion number 2."

I have a different explanation for why nitrogenase was recovered in LUCA. And this has to do with the tetrapyrrole biosynthesis enzymes related to nitrogenases that, in fact, do recover monophyly for Archaea and Bacteria. Namely, the enzyme involved in the synthesis of the Ni-tetrapyrrole cofactor, Cofactor F430, required for methanogenesis in archaea; and the enzymes involved in the synthesis of Mg-tetrapyrroles in photosynthetic bacteria. Still to this date, the subunits of the nitrogenase-like enzyme required for Cofactor F430 synthesis are annotated as nitrogenase subunits.

So, what Weiss et al interpreted as a nitrogenase in LUCA, might actually include proteins of the tetrapyrrole biosynthesis enzymes.

Bill, I think you should make all the trees for each one of the 355 proteins available online. That would be really useful for all of us interested in early evolution! Thank you.

On 2016 Oct 08, Shawn McGlynn commented:

This paper uses a phylogenetic approach to "illuminate the biology of LUCA" and uses two criteria to assess if a given protein encoding gene was in the LUCA:

"the protein should be present in at least two higher taxa of bacteria and archaea, respectively, and (2) its tree should recover bacterial and archaeal monophyly"

The authors later conclude that "LUCA accessed nitrogen via nitrogenase", however the nitrogenase protein is an excellent example of violating the author's criterion (2) above, and therefore cannot be included in the LUCA protein set based on the author's own criterion.

Upon phylogenetic analysis, the nitrogenase alpha subunit protein - which ligates the active site - branches into five clusters. One of these clusters is not well resolved, yet four of the five clusters contain both archaea and bacteria, therefor a nitrogenase protein phylogeny does not recover the monophyly of the archaea and bacteria.

Other claims in this paper may deserve scrutiny as well.

Suggested Reading below - if there are others to add someone please feel free:

Raymond, J., Siefert, J. L., Staples, C. R., and Blankenship, R. E. (2004). The natural history of nitrogen fixation. Mol. Biol. Evol. 21, 541–554

Boyd, E. S., Anbar, A. D., Miller, S., Hamilton, T. L., Lavin, M., and Peters, J. W. (2011a). A late methanogen origin for molybdenum-depen- dent nitrogenase. Geobiology 9, 221–232.

Boyd, E. S., Hamilton, T. L., and Peters, J. W. (2011b). An alternative path for the evolution of biological nitrogen fixation. Front. Microbiol. 2:205. doi: 10.3389/fmicb.2011.00205

On 2016 Oct 08, Shawn McGlynn commented:

This paper uses a phylogenetic approach to "illuminate the biology of LUCA" and uses two criteria to assess if a given protein encoding gene was in the LUCA:

"the protein should be present in at least two higher taxa of bacteria and archaea, respectively, and (2) its tree should recover bacterial and archaeal monophyly"

The authors later conclude that "LUCA accessed nitrogen via nitrogenase", however the nitrogenase protein is an excellent example of violating the author's criterion (2) above, and therefore cannot be included in the LUCA protein set based on the author's own criterion.

Upon phylogenetic analysis, the nitrogenase alpha subunit protein - which ligates the active site - branches into five clusters. One of these clusters is not well resolved, yet four of the five clusters contain both archaea and bacteria, therefor a nitrogenase protein phylogeny does not recover the monophyly of the archaea and bacteria.

Other claims in this paper may deserve scrutiny as well.

Suggested Reading below - if there are others to add someone please feel free:

Raymond, J., Siefert, J. L., Staples, C. R., and Blankenship, R. E. (2004). The natural history of nitrogen fixation. Mol. Biol. Evol. 21, 541–554

Boyd, E. S., Anbar, A. D., Miller, S., Hamilton, T. L., Lavin, M., and Peters, J. W. (2011a). A late methanogen origin for molybdenum-depen- dent nitrogenase. Geobiology 9, 221–232.

Boyd, E. S., Hamilton, T. L., and Peters, J. W. (2011b). An alternative path for the evolution of biological nitrogen fixation. Front. Microbiol. 2:205. doi: 10.3389/fmicb.2011.00205

On 2016 Oct 12, William F Martin commented:

There is an ongoing debate in the literature about the age of nitrogenase.

In his comment, McGlynn favours published interpretations that molybdenum nitrogenase arose some time after the Great Oxidation Event 2.5 billion years ago (1). A different perspective on the issue is provided by Stüecken et al. (2) who found evidence for Mo-nitrogenase before 3.2 billion years ago. Our recent paper (3) traced nitrogenase to LUCA, but also suggested that methanogens are the ancestral forms of archaea, in line both with phylogenetic (4) and isotope (5) evidence for the antiquity of methanogens, and with a methanogen origin of nitrogenase (6).

Clearly, there had to be a source of reduced nitrogen at life’s origin before the origin of nitrogenase or any other enzyme. Our data (3) are consistent with the view that life arose in hydrothermal vents and independent laboratory studies show that dinitrogen can be reduced to ammonium under simulated vent conditions (7,8). There is more to the debate about nitrogenase age, methanogen age, and early sources of fixed nitrogen than McGlynn’s comment would suggest.

1. Boyd, E. S., Hamilton, T. L., and Peters, J. W. (2011). An alternative path for the evolution of biological nitrogen fixation. Front. Microbiol. 2:205. doi:10.3389/fmicb.2011.00205

2. Stüeken EE, Buick R, Guy BM, Koehler MC. Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr. Nature 520, 666–669 (2015)

3. Weiss MC, Sousa FL, Mrnjavac N, Neukirchen S, Roettger M, Nelson-Sathi S, Martin WF: The physiology and habitat of the last universal common ancestor. Nat Microbiol (2016) 1(9):16116 doi:10.1038/nmicrobiol.2016.116

4. Raymann, K., Brochier-Armanet, C. & Gribaldo, S. The two-domain tree of life is linked to a new root for the Archaea. Proc. Natl Acad. Sci. USA 112, 6670–6675 (2015).

5. Ueno, Y., K. Yamada, N. Yoshida, S. Maruyama, and Y. Isozaki. 2006. Evidence from fluid inclusions for microbial methanogenesis in the early archaean era. Nature 440:516-519.

6. Boyd, E. S., Anbar, A. D., Miller, S., Hamilton, T. L., Lavin, M., and Peters, J. W. (2011). A late methanogen origin for molybdenum-depen- dent nitrogenase. Geobiology 9, 221–232.

7. Smirnov A, Hausner D, Laffers R, Strongin DR, Schoonen MAA. Abiotic ammonium formation in the presence of Ni-Fe metals and alloys and its implications for the Hadean nitrogen cycle. Geochemical Transactions 9:5 (2008) doi:10.1186/1467-4866-9-5

8. Dörr M, Kassbohrer J, Grunert R, Kreisel G, Brand WA, Werner RA, Geilmann H, Apfel C, Robl C, Weigand W: A possible prebiotic formation of ammonia from dinitrogen on iron sulfide surfaces. Angew Chem Int Ed Engl 2003, 42(13):1540-1543.

On 2016 Dec 25, Shawn McGlynn commented:

Unfortunately, the points raised by Professor Martin do not address the problem I raised in my original comment, which I quote from below: "nitrogenase protein phylogeny does not recover the monophyly of the archaea and bacteria." As I wrote, the nitrogenase protein is an excellent example of violating the author's criterion of judging a protein to be present in the LUCA by virtue that "its tree should recover bacterial and archaeal monophyly" (quoted from Weiss et alia). Therefore it should not be included in this paper's conclusions.

Let's be more specific about this and look at a phylogenetic tree of the nitrogenase D peptide (sometimes referred to as the alpha subunit). This peptide binds the catalytic metal-sulfur cluster and its phylogeny can be viewed on my google site https://sites.google.com/site/simplyshawn/home.

I colored archaeal versions red and bacterial black. You can see that this tree does not recover the monophyly of the archaea and bacteria and therefore should not be included in the author's set of LUCA proteins.

Is what I display the result of a tree construction error? Probably not, this tree looks pretty much the same to every other tree published by various methods, so it seems to correctly reflect sequence evolution as we understand it today. The tree I made just has more sequences; it can be compared directly with Figure 1 in Leigh 2000, Figure 2 in Raymond et alia 2004, and Figure 2 in Boyd et alia 2011. Unfortunately, Weiss and others do not include any trees in their paper, so it is impossible to know what they are deriving their conclusions from, but it would be very difficult to imagine that they have constructed a tree different from all of these.

Could it be that all these archaea obtained nitrogenase by horizontal gene transfer after the enzyme emerging in bacteria? Possibly, although this would imply that it was not in the LUCA as the authors claim.

Could it be that the protein developed in methanogens and was then transferred into the bacterial domain? Yes, and Boyd and others suggested just this in their 2011 Geobiology paper. This would also mean that the protein was not in the LUCA.

Could it be that the protein was present in the LUCA as Weiss and co-authors assert? Based on phylogenetic analysis, no.

As Prof. Martin writes - there certainly is more debate to be had about nitrogenase age than was visible in my first comment. However, we can be sure that the protein does not recover the archaea bacteria monophyly, and should have not been included in the authors paper.

Prof. Martin might likely counter my arguments here by saying something about metal dependence and treating different sequences separately (for example Anf, Vnf, MoFe type). However let us remember that the sequences are all homologous. Metal binding is one component of the nitrogenase phenotype, but all nitrogenase are homologous and descend from a common ancestor.

Now that we can be sure that the nitrogenase does not conform to the author's second criterion for judging presence in the LUCA, let us examine if the protein conforms to the first criterion: "the protein should be present in at least two higher taxa of bacteria and archaea". In fact, all nitrogenase in archaea that are found in the NCBI and JGI databases are only within the methanogenic euryarchaeota. Unfortunately, Weiss and coauthors do not define what "higher taxa" means to them in their article, but it should be questioned if having a gene represented by members of a single phylum actually constitutes being present within "two higher taxa". Archaea are significantly more diverse than what is observed in the methanogenic euryarchaeota. Surely, if a protein was present in the LUCA, it would be a bit more widely distributed, and it would be easy to argue that the presence of nitrogenase in only one phylum provides evidence that it does not conform to the authors criterion number one. Thus, the picture that emerges from a closer look at nitrogenase phylogeny and distribution is that the protein violates both of the authors criteria for inclusion in the LUCA protein set.

Let me summarize:

1) Nitrogenase does not recover the bacterial and archaeal monophyly and therefore violates the author's criterion number 2.

2) Nitrogenase in archaea is only found within the methanogenic euryarchaeota and is not broadly distributed, and therefore also seems to violate the authors criterion number 1.

3) From a phylogenetic perspective, the nitrogenase protein should not be included as a candidate to be present in the LUCA.

On 2017 Mar 20, Madeline C Weiss commented:

The trees and also the alignments for all 355 proteins are available on our resources website:

http://www.molevol.de/resources/index.html?id=007weiss/

On 2017 Jun 10, Shawn McGlynn commented:

With the trees from this phylogeny paper now available, we can resolve the discussion between myself and the authors (below) and conclude that there is no evidence that nitrogenase was present in the LUCA as the authors claimed in their publication.

In their data set, the authors identified two clusters of proteins which they refer to as NifD; clusters 3058 and 3899. NifD binds the metal cluster of nitrogenase and is required for catalysis. In the author's protein groups, cluster 3058 is comprised of 30 sequences, and 3899 is comprised of 10 sequences. Inspection of these sequences reveals that neither cluster contains any actual NifD sequences. This can be said with certainty since biochemistry has demonstrated that the metal cofactor coordinating residues Cys²⁷⁵ and His⁴⁴² (using the numbering scheme from the Azotobacter vinelandii NifD sequence) are absolutely required for activity. NONE of the 40 sequences analyzed by the authors contain these residues. Therefore, NONE of these sequences can have the capability to bind the nitrogenase metal cluster, and it follows that none of them would have the capacity to reduce di-nitrogen. The authors have not analyzed a single nitrogenase sequence in their analysis and are therefore disqualified from making claims about the evolution of the protein; the claims made in this paper about nitrogenase cannot be substantiated with the data which have been analyzed. The sequences contained in the author's "NifD" protein clusters are closely related homologs related to nitrogenase cofactor biosynthesis and are within a large family of related proteins (which includes real NifD proteins, but also proteins involved in bacteriochlorophyll and Ni porphyrin F430 biosynthesis). While the author's analyzed proteins are more related to nitrogen metabolism than F430 or bacteriochlorophyll biosynthesis, they are not nitrogenase, but are nitrogenase homologs that complete assembly reactions.

Other than not having looked at any sequences which would be capable of catalyzing nitrogen reduction, the presentation of two "NifD" clusters highlights important problems with the methods used in this paper which affect the entire analysis and conclusions. First, two clusters were formed for one homologous group, which should not have occurred if the goal was to investigate ancestry. Second, by selecting small clusters from whole trees, the authors were able to prune the full tree until they recovered small sub trees which show monophyly of archaea and bacteria. However it was incorrect to ignore the entire tree of homologs and present only two small clusters from a large family. This is "cherry" picking to the extreme - in this case it is "nitrogenase" picking, but it is very likely that this problem of pruning until the desired result sullies many if not all of the protein families and conclusions in the paper; for example the radical SAM tree was likely pruned in this same way with the incorrect conclusion being reached (like nitrogenase, a full tree of radical SAM does not recover the archaea bacteria split in protein phylogenies either). Until someone does a complete analysis with full trees the claims of this paper will remain unproven and misleading since they are based on selective sampling of information. It would seem that the authors have missed the full trees whilst being lost in mere branches of their phylogenetic forest of 286,514 protein clusters.

In a forthcoming publication, I will discuss in detail the branching position of the NifD homologs identified by the authors, as well as the possible evolutionary trajectory of the whole protein family with respect to the evolution of life and the nitrogen cycle on this planet in more detail, including bona fide NifD proteins which I have already made comment on below in this PubMed Commons thread.